Compositions, methods and uses for thermally stable multi-targeted antigens

ABSTRACT

Embodiments of the present invention provide for novel compositions and methods for making and using a thermally stable human papilloma virus (HPV) formulation or other stabilized multimeric virus formulation. Certain embodiments concern lyophilizing HPV formulations in the presence or absence of adjuvants. Other embodiments concern lypophilizing HPV capsomere vaccines in order to increase stability of an immunogenic composition against HPV infection for storage, delivery and use. In yet other embodiments, a single immunogenic composition can include a thermally stable formulation of multiple virus serotypes. Yet other embodiments disclosed herein concern multi-targeted antigen complexes lyophilized in formulations of use to prolong stability and/or enhance immunogenicity. Other embodiments concern exposing lyophilized multi-targeted antigen complexes to elevated temperatures to enhance immunogenicity of the antigens of the complex to multiple pathogens.

PRIORITY

This Continuation Application is a Continuation of application Ser. No.16/146,686, filed Sep. 28, 2018, allowed, which is aContinuation-in-Part Application that claims priority to U.S. 371application Ser. No. 15/309,169 filed Nov. 4, 2016, now abandoned, whichclaims priority to PCT Application No. PCT/US2015/029529 filed May 6,2015 which claims the benefit of U.S. Provisional Application Ser. No.61/989,365 filed May 6, 2014. These applications are incorporated hereinby reference in their entirety for all purposes.

FIELD

Embodiments of the present invention provide for novel compositions andmethods for making and using a thermally stable human papilloma virus(HPV) vaccine or immunogenic formulation or other stabilized multimericvirus vaccine or immunogenic formulation. Certain embodiments concernlyophilizing HPV formulations in the presence or absence of adjuvants.Other embodiments concern lypophilizing HPV capsomere vaccines and otherimmunogenic agents to increase stability or reduce degradation of thevaccine and/or agents for storage, delivery and use. In yet otherembodiments, a single immunogenic formulation can include a thermallystable composition of multiple virus serotypes. Certain embodimentsconcern lyophilizing multi-targeted antigen complexes in the presence ofvarious agents to increase stability or reduce degradation of antigenicagents prolonging storage stability, delivery and use. In yet otherembodiments, a single immunogenic formulation can include a thermallystable composition of a broad-spectrum multi-targeted antigeniccomposition against multiple pathogens. In some embodiments, astabilizing formulation can include a hypertonic mixture including oneor more disaccharide and one or more volatile salts for lyophilizationand prolonged storage of the multi-targeted antigens (e.g. RG1 HPV16VLP)or the like. In yet another embodiment, exposure to elevatedtemperatures of a stabilized, lyophilized multi-targeted antigen complexdisclosed herein can increase cross-reactivity of the complex againstmultiple pathogens compared to a control when reconstituted andintroduced to a subject.

BACKGROUND

Papillomaviruses infect a wide variety of different species of animalsincluding humans. Infection is typically characterized by the inductionof benign epithelial and fibro-epithelial tumors, or warts at the siteof infection. Each species of vertebrate is infected by aspecies-specific set of papillomaviruses, including several differentpapillomavirus types. For example, more than one hundred different humanpapillomavirus (HPV) genotypes have been isolated. Papillomaviruses arehighly species-specific infective agents. For example, canine and rabbitpapillomaviruses cannot induce papillomas in heterologous species suchas humans. Neutralizing immunity to infection against one papillomavirustype generally does not confer immunity against another type, even whenthe types infect a homologous species.

In humans, papillomaviruses can cause genital warts, a prevalentsexually-transmitted condition. HPV types 6 and 11 are most commonlyassociated with benign genital warts (e.g., condylomata acuminate).Genital warts are very common, and subclinical or unapparent HPVinfection is even more common than clinical infection. While mostHPV-induced lesions are benign, lesions arising from certainpapillomavirus types e.g., HPV-16 and HPV-18, can undergo malignantprogression. Moreover, infection by one of the malignancy-associatedpapillomavirus types is considered to be a significant risk factor inthe development of cervical cancer. Cervical cancer is the third mostcommon cancer in women worldwide. Most cervical cancer cases occur inwomen living in developing countries where availability of vaccines andpreventative screenings, such as pap smears are limited. HumanPapillomavirus (HPV) is the etiologic agent associated with cervicalcancer, and therefore vaccines against HPV would be very beneficial inreducing the disease prevalence in developing countries.

Delivering an effective HPV vaccine or other multi-targeted antigeniccomplex compositions to developing countries comes with many challenges.Ideally, the cost of a (e.g., HPV) vaccine for developing countriesneeds to be inexpensive as possible. Additionally, keeping vaccines at atemperature sufficient to maintain the composition and reducedegradation can be difficult when delivering vaccines to remote regionsand limited refrigerated space is available for vaccine storage. Therecommended temperature ranges for transporting vaccines inrefrigeration or cooler temperatures are narrow. If liquid vaccineformulations are exposed to freezing or elevated temperatures,degradation or loss of efficacy can result. Limitations of maintaining avaccine in refrigerated storage are even more pronounced when deliveringthe vaccine in a developing country.

HPV-16 is the most common of the HPV genotypes involved in cervicalcancer making up about 50% of cervical cancers. Prevalence of HPV-18ranges from approximately 8-31% of cervical cancers depending on thegeographical location. HPV-45 is the third most frequent oncogenic HPVtype. Other cancer-related genotypes include HPV-31, HPV-33, HPV-52,HPV-58, HPV-35, HPV-59 and HPV-56. One of the issues involved with theproduction and use of HPV vaccines has been effective in providingeffective storage and transportation of the vaccines where storageconditions can reduce degradation or increase stability of a viralvaccine formulation.

SUMMARY

Embodiments of the present invention provide for novel compositions andmethods for making and using a thermally stable human papilloma virus(HPV) formulation or other stabilized multimeric virus formulation.Certain aspects concern partially or fully lyophilizing or freeze-dryingHPV formulations in the presence or absence of one or more adjuvants orother immune-stimulating agents. Other embodiments described hereinconcern lypophilizing HPV capsomere vaccines or freeze-drying HPVcapsomeres constructs to increase stability or decrease degradation ordisassembly of the vaccines or constructs during storage,transportation, delivery and use.

In some embodiments, lyophilized glassy-state HPV vaccines can bedeveloped using any HPV antigen in combination with an adjuvant. Incertain embodiments, HPV-16 and HPV-18 as well as HPV-31, HPV-33,HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-6, HPV-11, HPV-30,HPV-42, HPV-43, HPV44, HPV-54, HPV-55, and HPV-70 are contemplated ofuse herein. In other embodiments, lyophilized glassy-state HPV vaccinescan be developed using HPV L1 capsomere proteins as an antigen combinedwith an adjuvant. Adjuvants contemplated herein include, but are notlimited to, aluminum hydroxide or aluminum hydroxide withglycopyranoside lipid A (GLA). In some embodiments, an adjuvant caninclude an aluminum salt including but not limited to, one or more ofaluminum hydroxide, aluminum phosphate and aluminum sulfate, orcombinations thereof. In other embodiments, the aluminum salt can be inthe form of an aluminum hydroxide gel (e.g., ALHYDROGEL). Otheradjuvants contemplated herein include, but are not limited to,calcium-based salts including calcium phosphate, muramyl dipeptide,oligodeoxynucleotides containing CpG motifs, bacterial flagellins,saponins such as Quils. ISCOM and QS21, resquimod, MF 59 emulsions,squalene emulsions, cytokines such as IL-2, IL-12 and GMCSF, silica,polynucleotides, toxins, such as cholera toxin, toxoids, such as choleratoxoid, serum proteins, other viral coat proteins, otherbacterial-derived preparations, block copolymer adjuvants, such asHunter's TITERMAX adjuvant (VAXCEL, Inc., Norcross, Ga.); RIBI adjuvants(available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.),liposomes, and microparticles of polymers such as poly-(lactic acid) andpoly-(lactic-co-glycolic acid).

In certain aspects of the invention, vaccine formulations can belyophilized for example, where an L1 pentamer remains intact. Inaddition, these combinations can reduce detrimental modifications tocritical neutralizing epitopes of the L1 pentamer. In other embodiments,HPV vaccines or compositions disclosed herein preserved antibody titerby increasing stability and/or decreasing disassembly or degradation. Inother embodiments, the antigen compositions described herein can reduceantibody titer loss at temperatures of about 40° C. to about 50° C. toabout 60° C. degrees for up to several weeks to months making itpossible to store and transport vaccine compositions at an increasedtemperature for a longer duration. It is anticipated that theseprinciples can be applied to other vaccine formulations, includingvaccines formulated with virus-like particles, vaccine formulationscontaining live, attenuated viruses and vaccines containing proteinantigens can all benefit from the compositions and methods disclosedherein.

In other embodiments, vaccine compositions of the instant invention canbe used to vaccinate subjects in order to reduce consequences of a viralinfection or potentially prevent infection and side effects of a viralinfection. For example, compositions of HPV 16 L1 capsomere proteins incombination with adjuvants can be lyophilized and transported to remoteareas for distribution and administration to subjects in need. In otherembodiments, vaccine formulations described herein can be used alone orin combination with other agents used to prevent HPV infections in asubject (e.g., GARDASIL and CERVARIX).

In other embodiments, vaccine or immunogenic compositions disclosedherein can contain multiple types of HPV L1 capsomeres that can be usedto immunize or vaccinate subjects in need thereof. In accordance withthese embodiments, compositions of mixtures of HPV 16 L1 capsomeres, HPV18 LI capsomeres, HPV31 capsomeres and/or HPV 45 capsomeres can beco-lyophilized and transported to remote areas for distribution andimmunization of subjects in need. In certain embodiments, variouscombinations of any HPV L1 capsomeres can be combined with adjuvants andco-lyophilized and transported to remote areas for distribution andimmunization of subjects in need.

In other embodiments, vaccine or immunogenic compositions disclosedherein can contain multimeric compositions of HPV16 L1, HPV18 L1, HPV 31L1, and HPV45 L1 capsomeres, for example. In accordance with theseembodiments, immunogenic compositions disclosed herein can also containparticulate adjuvants such as aluminum or aluminum salt adjuvants, forexample aluminum hydroxide or aluminum hydroxide with glycopyranosidelipid A (GLA), as well as glass-forming agents, such as trehalose and/orsucrose. In some embodiments, these immunogenic compositions can beco-lyophilized, stored and/or transported to remote areas where they canbe reconstituted with no loss of multimeric structure or immunogenicity.

Certain embodiments provide for novel compositions and methods for athermally stable broad-spectrum multi-targeted antigen formulation. Someaspects concern partially or fully lyophilizing or freeze-drying thebroad-spectrum multi-targeted antigen formulation in the presence of ahypotonic mixture. Other embodiments described herein concernlyophilizing broad-spectrum multi-targeted antigen constructs (e.g., RG1HPV16VLPs) to increase stability or decrease degradation or disassemblyof the constructs during storage, transportation and delivery resultingin a reduction of product loss and reduction of loss of efficacy.

In some embodiments, broad spectrum multi-targeted antigens can belyophilized and dried to create powdered formulations. In certainembodiments, constructs can include RG1 HPV16VLPs or similar (U.S. Pat.No. 9,149,503 is incorporated herein in its entirety for all purposes).In other embodiments, multi-targeted antigen complexes can belyophilized and dried to create a powdered formulation subjected toelevated temperatures (e.g., 40-60° C.) then reconstituted to enhance animmune response in a subject to the targets represented by themulti-targeted antigen and to enhance cross-reactivity.

In certain embodiments, compositions disclosed herein include, but arenot limited to, one or more volatile salts. In accordance with theseembodiments, one or more volatile salts can include, but are not limitedto, one or more of ammonium acetate, ammonium formate, ammoniumcarbonate, ammonium bicarbonate, triethylammonium acetate,triethylammonium formate, triethylammonium carbonate, trimethylamineacetate trimethylamine formate, trimethylamine carbonate, pyridinalacetate and pyridinal formate, or combinations thereof.

In other embodiments, formulations of use herein can include one or morenon-reducing disaccharides including, but not limited to, trehalose,sucrose and lactose, and optionally, additional glass-forming agents.Glass-forming agents can include, but are not limited to, hydroxyethylstarch, glycine, glycine and mannitol, cyclodextrin, and polyvinylpyrrolidone (povidone) or combinations thereof.

In some embodiments, formulations of use herein can include amulti-targeted antigen (e.g., VLP assembled from an HPV L1 protein), oneor more disaccharide and one or more volatile salt or volatile saltbuffer. In accordance with these embodiments, a multi-targeted antigencan be a complex made up of antigens derived from several pathogens(e.g. immunogenic epitopes), a non-reducing disaccharide can include oneor more of trehalose, sucrose, lactose, or the like and one or morevolatile salts can include one or more of ammonium acetate, ammoniumformate, ammonium carbonate, ammonium bicarbonate, triethylammoniumacetate, or the like. In certain embodiments, a stabilizing formulationof use to prolong shelf life of a multi-targeted antigen, such as RG1HPV16VLPs or similar constructs or other multi-faceted antigen complexescan include a hypertonic mixture including trehalose and ammoniumacetate.

In certain aspects of the instant disclosure, immunogenic formulationsof broad-spectrum multi-targeted antigen formulations can be lyophilizedfor example, where the broad-spectrum construct remains intact. Inaddition, these combinations can reduce detrimental modifications tocritical neutralizing epitopes of an assembled antigen. In otherembodiments, broad-spectrum multi-targeted antigen compositions canpreserve antibody titer by increasing stability and/or decreasingdisassembly or degradation. In other embodiments, multi-targeted antigencompositions described herein can be stabilized to reduce antibody titerloss when stored at temperatures of about 40° C. to about 50° C. toabout 60° C. degrees for up to several weeks to several months making itpossible to store and transport without product loss for a longerduration. In certain embodiments, immunogenic compositions ofmulti-targeted antigens, (e.g., RG1 HPV16VLPs) or the like can be frozenon precooled shelves of a lyophilizer and dried under vacuum creating anessentially dry powder formulation. In other embodiments, theseconstructs can be combined with trehalose and ammonium acetatelyophilized and dried to prolong shelf-life of the active agents.

In yet other embodiments, these stored formulations can be stored atelevated temperatures (at about 40 to about 60° C.) and subsequentlyreconstituted for use against multiple pathogens. In certainembodiments, the multi-targeted antigen can be stored at elevatedtemperatures (at about 40 to about 60° C.) for a few hours, to one day,a week or month or more prior to reconstitution to enhance crossreactivity of the multi-targeted antigen complex.

In other embodiments, vaccine or immunogenic compositions disclosedherein can contain broad-spectrum constructs. In accordance with theseembodiments, immunogenic compositions disclosed herein can also containparticulate adjuvants such as aluminum or aluminum salt adjuvants, forexample aluminum hydroxide or aluminum hydroxide with glycopyranosidelipid A (GLA), as well as disaccharide agents, such as trehalose and/orsucrose. In some embodiments, these immunogenic compositions can beco-lyophilized, stored and/or transported to remote areas where they canbe reconstituted with no loss of multimeric structure or immunogenicity.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the instant specification and areincluded to further demonstrate certain aspects of particularembodiments herein. The embodiments may be better understood byreference to one or more of these drawings in combination with thedetailed description presented herein.

FIGS. 1A-1C are photographic representations of electron microscopeimages of certain embodiments presented herein, before lyophilization(A), immediately after lyophilization and reconstitution (B), and afterstorage in the lyophilized state and reconstituted (C).

FIG. 2 represents an exemplary histogram plot of Stern-Volmer constantsfor time 0 vaccine formulations of certain embodiments presented herein.

FIGS. 3A and 3B are graphical representations of HPV 16 L1 capsomerereactivity to V5 (A) and L 1(B) antibodies measured using absorbance at450 nm, according to one embodiment of the present disclosure.

FIG. 4 represents histogram plots of particle size and concentration ofHPV vaccines under various storage conditions, according to certainembodiments herein.

FIGS. 5A and 5B represent graphic illustrations of anti-HPV-16antibodies (A) and neutralizing antibodies (B) after one (solid circles)and two (open circles) vaccine injections for protein (P), protein+alum(PA), protein+alum+GLA (PAG), GARDASIL, and CERVARIX vaccines.

FIGS. 6A and 6B represent a graphic illustration of time 0 and incubatedvaccines using anti-HPV-16 antibodies (A) and neutralizing antibodies(B) after one (solid circles) and two (open circles) vaccine injectionsfor protein (P), protein+alum (PA), protein+alum+GLA (PAG), GARDASIL,and CERVARIX vaccines.

FIGS. 7A and 7B represent graphic illustrations of dose response curvesfor antibody neutralization studies (A) and antibody incubation study inmice (B) for certain vaccine compositions according to embodimentsdisclosed herein.

FIGS. 8A and 8B are photographic representations of electron microscopetransmissions of certain embodiments presented herein, beforelyophilization (A), and after lyophilization and reconstitution (B) ofHPV16 L1 capsomeres.

FIGS. 9A and 9B are photographic representations of electron microscopetransmissions of certain embodiments presented herein, beforelyophilization (A), and after lyophilization and reconstitution (B) ofHPV18 L1 capsomeres.

FIGS. 10A and 10B are photographic representations of electronmicroscope transmissions of certain embodiments presented herein, beforelyophilization (A), and after lyophilization and reconstitution (B) ofHPV31 L1 capsomeres.

FIGS. 11A and 11B are photographic representations of electronmicroscope transmissions of certain embodiments presented herein, beforelyophilization (A), and after lyophilization and reconstitution (B) ofHPV45 L1 capsomeres.

FIG. 12 is a photographic representation of an electron microscopetransmission of an embodiment presented herein, after lyophilization andreconstitution of a tetravalent formulation containing HPV16 L1, HPV18L1, HPV31 L1, and HPV45 L1 capsomeres.

FIGS. 13A-13C are photographic representations of SDS Page and WesternBlot analysis of certain embodiments presented herein, beforelyophilization (A), and after lyophilization and reconstitution (B) ofHPV16 L1, HPV18 L1, HPV31 L1, and HPV45 L1 capsomeres, and (C) beforelyophilization (“PRE”) and after lyophilization and reconstitution(“POST”) of a tetravalent formulation containing HPV16 L1, HPV18 L1,HPV31 L1, and HPV45 L1 capsomeres.

FIG. 14 represents a table reflecting exemplary data where titer of aconstruct of various embodiments disclosed herein was measured atindicated temperatures over time.

FIGS. 15A and 15B represents some data obtained from an exemplaryneutralization assay to detect neutralization data against HPV16 as wellas some cross neutralization data against other antigens of certainembodiments disclosed herein.

FIG. 16 represents a histogram plot of a mouse model testing stimulationof T cell responses after immunization with a broad-spectrum HPV complexstored in exemplary compositions at various temperatures and times whenthe mice are exposed to various agents including positive and negativecontrols.

FIG. 17 represents an image of an exemplary scanning electron micrographof RGI-HPV VLPs demonstrating intact VLPs in certain embodimentsdisclosed herein.

DEFINITIONS

In order to facilitate an understanding of the invention, the followingdefinitions are provided.

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, “about” may mean up to and including plus or minus fivepercent, for example, about 100 may mean 95 and up to 105.

Capsid protein: the structural protein of a virus, e.g., enveloped, ornon-enveloped, which constitutes the capsid structure. Generally, thereare several capsid proteins which are often described by whether theyare the predominant (major) constituent or lesser (minor) constituent ofcapsid structure.

Conformational antibody: refers to an antibody that specifically bindsan epitope expressed as a correctly-folded L1 or L2 protein but not ondenatured L1 or L2 protein.

Capsomere: refers to a structure that makes up the larger viral capsidstructure that is generally a pentamer of one type of capsid proteins.In the case of HPV, a native capsomere comprises a pentamer of L1 capsidproteins that may be associated with one L2 capsid protein.

“Capsid” as used herein refers to the structural portion of a virus,e.g., HPV that is comprised of capsomeres. In the case of HPV, the viralcapsid is comprised of 72 capsomeres.

“Chimeric protein” as used herein refers to a protein created when twoor more genes that normally code for two separate proteins recombine,either naturally or as the result of human intervention, to code for aprotein that is a combination of all or part of each of those twoproteins.

“Multi-targeted antigen” as used herein refers to an antigen complexwhere the antigen can be derived from bacteria, viruses, fungi, orsegments, peptides, epitopes derived thereof. For example, as usedherein a multi-targeted antigen can be a single complex capable ofeliciting multiple protective immunogenic responses at the same time,for example, simultaneously.

DETAILED DESCRIPTIONS

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments. It will be obviousto one skilled in the art that practicing the various embodiments doesnot require the employment of all or even some of the details outlinedherein, but rather that concentrations, times and other details may bemodified through routine experimentation. In some cases, well knownmethods or components have not been included in the description.

In certain embodiments, compositions, methods and uses for stabilizingHPV vaccine formulations are disclosed. A formulation or application ofa formulation that can stabilize viral vaccines from for example, fromdegradation or dissolution of a viral structure is disclosed. In certainembodiments, compositions disclosed herein can be used to reduce loss oftiter of lyophilized HPV formulations. In certain embodiments,compositions disclosed herein can concern a combination of two or moreagents (e.g., adjuvant or adjuvant-like agent) provided to an HPVvaccine formulation where the formulation is then lyophilized.

In some embodiments, vaccine formulations can be lyophilized in thepresence of glass-forming excipients, and sufficient liquid can beremoved during lyophilization that the dried or essentially driedvaccine formulation or immunogenic composition exhibits a glasstransition temperature that is higher than the anticipated storagetemperature. For example, the anticipated storage temperature may beroom temperature.

In certain embodiments, one or more agents provided to a vaccine orimmunogenic formulation disclosed herein can include, but is not limitedto, one or more aluminum-salt adjuvants, one or more buffer systemscontaining one or more one volatile salts, one or more one glass-formingagents, one or more immunologically-related co-stimulatory agents andone or more multimeric protein antigens. In certain aspects, aformulation can be combined to create a liquid vaccine or immunogenicformulation. In other aspects, an immunogenic or vaccine formulation canbe frozen to create a frozen immunogenic or vaccine formulation. In yetother aspects, the vaccine formulation or immunogenic formulation can belyophilized to create a dried or essentially dried vaccine orimmunogenic composition. In yet other embodiments, the viruscompositions disclosed herein can go through a classification step inthe presence of one or more adjuvants. Certain embodiments disclosedherein concern incubation of lyophilized multi-targeted antigencomplexes exposed for prolonged periods at elevated temperatures toenhance immune response to the multiple targets that make up themulti-targeted antigen and induce enhanced cross-reactivity to varioustypes or serotypes of pathogenic organisms such as viruses, bacteria,fungi or the similar (e.g, flaviviruses, alphaviruses etc.). In oneexemplary embodiment, a complex contemplated in formulations and methodsdisclosed herein can include a RG1 HPV-VLP or similar.

In some embodiments, a multimeric viral protein complex as part of avaccine or immunogenic composition can include one or more capsomeresformed from proteins derived from a viral capsid. For example, amultimeric viral protein can include a pentamer assembled from LIproteins of the human papilloma virus. In some embodiments, a multimericviral protein is an HPV 16 L1 capsomere. In other embodiments, amultimeric viral protein can include capsomeres of HPV18 L1 protein,HPV31 L1 protein or HPV45 L1 protein, alone or in combination with HPV16 L1. In other embodiments, a multimeric viral protein is another HPVcomplex such as a virus-like particle (VLP) or other viral complex withsimilar characteristics to a capsomere wherein the glassy excipientsdisclosed herein stabilize the viral complex when stored or transportedat increased temperatures avoiding the need for long-term refrigeration.

In other embodiments, vaccine or immunogenic compositions disclosedherein can contain multimeric compositions of HPV16 L1, HPV18 L1, HPV 31L1, and HPV45 L1 capsomeres, for example. In accordance with theseembodiments, immunogenic compositions disclosed herein can also containparticulate adjuvants. In certain embodiments, particulate adjuvants canbe aluminum or aluminum salt adjuvants, for example aluminum hydroxideor aluminum hydroxide with glycopyranoside lipid A (GLA). In otherembodiments, these compositions can include glass-forming agents. Glassforming agents can include but are not limited to, trehalose, sucrose,raffinose, ficoll, dextran, sucrose, maltotriose, lactose, mannitol,hydroxyethyl starch, glycine, cyclodextrin, and polyvinyl pyrrolidone(povidone).

In some embodiments, these immunogenic compositions can beco-lyophilized, stored and/or transported to remote areas where they canbe reconstituted with no loss of multimeric structure or immunogenicity.

In some embodiments, the aluminum salt adjuvant of the vaccinecomposition can include one or more of aluminum hydroxide, aluminumphosphate and aluminum sulfate, or combinations thereof. In otherembodiments, the aluminum salt can be in the form of an aluminumhydroxide gel (e.g., ALHYDROGEL) or other consistency.

In certain embodiments, a buffer of use in compositions disclosed hereincan include, but is not limited to, one or more volatile salts. Inaccordance with these embodiments, one or more volatile salts caninclude, but are not limited to, one or more of ammonium acetate,ammonium formate, ammonium carbonate, ammonium bicarbonate,triethylammonium acetate, triethylammonium formate, triethylammoniumcarbonate, trimethylamine acetate trimethylamine formate, trimethylaminecarbonate, pyridinal acetate and pyridinal formate, or combinationsthereof.

In other embodiments, a glass-forming agent (e.g., when freeze-dried thecompositions forms a glass-like consistency instead of crystals)disclosed herein can include one or more of trehalose, sucrose, ficoll,dextran, sucrose, maltotriose, lactose, mannitol, hydroxyethyl starch,glycine, cyclodextrin, and povidone, or combinations thereof. In someembodiments, the glass-forming agent in a weight-to-volume (w/v)concentration of from about 1% to about 20%, or about 5% to about 15% ina liquid vaccine formulation prior to lyophilization. In otherembodiments, the glass-forming agent can be trehalose present in aconcentration of from about 8% to about 20% w/v in the liquid vaccineformulation prior to lyophilization. In another embodiment, theglass-forming agent can be trehalose at a concentration of about 9.5%w/v in the liquid vaccine formulation or immunogenic composition priorto lyophilization. Glass-forming agents that can be used in accordancewith the various embodiments of the present disclosure can include, butare not limited to, trehalose, sucrose, ficoll, dextran, sucrose,maltotriose, lactose, mannitol, hydroxyethyl starch, glycine,cyclodextrin, polyvinyl pyrrolidone, and the like.

In some embodiments, compositions disclosed herein can include both abuffer composed of volatile salts and a glass forming agent atconcentrations that are hypertonic prior to lyophilization, but that asa result of buffer volatilization during the lyophilization processbecome isotonic upon reconstitution.

In some embodiments, a co-stimulatory agent of a vaccine or immunogeniccomposition disclosed herein can include one or more of lipid A, lipid Aderivatives, monophosphoryl lipid A, chemical analogues ofmonophosphoryl Lipid A, CpG containing oligonucleotides, TLR-4 agonists,flagellin, flagellins derived from gram negative bacteria, TLR-5agonists, fragments of flagellins capable of binding to TLR-5 receptors,saponins, analogues of saponins, QS-21, purified saponin fractions,ISCOMS and saponin combinations with sterols and lipids, or combinationsthereof. In some embodiments, the co-stimulatory agent can be about 0.05mg/mL Glycopyranoside lipid A (GLA).

In some embodiments, a vaccine composition can be formulated to includeabout 0.1 mg/mL HPV 16 L1 capsomere, about 0.5 mg aluminum hydroxide gel(e.g., ALHYDROGEL), about 0.05 mg/mL Glycopyranoside lipid A (GLA) in 54mM histidine HCl (pH about 7.1), and about 9.5 w/v % trehalose.

In some embodiments, stability of vaccine or immunogenic compositionsdisclosed herein can be enhanced by the addition of nonionicsurfactants. In accordance with these embodiments, surfactants can beadded to vaccine or immunogenic formulations at concentrations rangingfrom approximately 0.1 times the critical micelle concentration of thesurfactant in the vaccine composition, to approximately 20 times thecritical micelle concentration of the surfactant in the vaccinecomposition before, during or after lyophilization of the composition.Suitable nonionic surfactants include, but are not limited to,polysorbates such as Tween 20, Tween 40, Tween 60 and Tween 80,poloxamers for example Polaxamer 188 and Poloxamer 407, Poloxamer 235,Poloxamer 335, Brij, alkylphenol hydroxypolyethylene surfactants such asTriton X100, Triton X114 and Triton X405, and Oligoethylene glycolmonoalkyl ethers such as Genapol.

In certain embodiments, compositions, methods and uses for stabilizingmulti-targeted antigen formulations are disclosed. A formulation orapplication of a formulation that can stabilize antigenic vaccinecomplexes from; for example, from degradation or dissolution of a viralstructure is contemplated. In certain embodiments, compositionsdisclosed herein can be used to reduce loss of titer of lyophilizedmulti-targeted antigen formulations (e.g., HPV). In other embodiments,compositions disclosed herein can concern a combination of two or moreagents (e.g., adjuvant or adjuvant-like agent) provided to amulti-targeted antigenic formulation where the formulation is thenlyophilized.

In certain embodiments, a multi-targeted antigen contemplated herein caninclude antigens derived from two or more pathogenic organisms. Inaccordance with these embodiments, a multi-targeted antigen can includeantigens from multiple species or multiple pathogens complexed to for amulti-targeted antigen. For example, a chimeric viral complex, liveattenuated virus complexes, multi-peptide cytomegalovirus (CMV)-modifiedvaccinia Ankara (MVA) vaccine, Plasmodium falciparum multiple-antigenpeptide vaccines, PnuBioVax (PBV multi-antigen, serotype-independentprophylactic vaccine against S. pneumoniae disease, ALVAC(2), melanomamulti-antigen therapeutic vaccine, bacterial backed complexes (e.g.salmonella, MVA constructs), flavivirus antigenic complexes, alphavirusantigenic complexes are contemplated herein.

In some embodiments, vaccine formulations can be lyophilized in thepresence of one or more disaccharide and one or more volatile salt andsufficient liquid can be removed during lyophilization that the dried oressentially dried vaccine formulation or immunogenic composition isstabilized from degradation. In other embodiments, these complexes canbe stored for one day, one week, one month or more. One anticipatedstorage temperature of lyophilized complexes disclosed herein can beroom temperature or higher (e.g. about 30° C. to about 60° C.).

Embodiments of the present invention provide for novel compositions andmethods for a thermally stable broad-spectrum multi-targeted antigenformulation. Certain aspects concern partially or fully lyophilizing orfreeze-drying the broad-spectrum multi-targeted antigen formulation inthe presence of a hypotonic mixture. Other embodiments described hereinconcern lyophilizing broad-spectrum constructs (e.g. RG1 HPV16VLPs) toincrease stability or decrease degradation or disassembly of theconstructs during storage, transportation, delivery resulting in areduction of product loss and reduction of loss of efficacy.

In some embodiments, broad spectrum multi-targeted antigens arelyophilized and dried to create powdered formulations. In certainembodiments, constructs can include RG1-VLPs, RG1 HPV16VLPs or similar(U.S. Pat. No. 9,149,503 is incorporated herein in its entirety for allpurposes).

In certain embodiments, compositions disclosed herein include, but arenot limited to, one or more volatile salts. In accordance with theseembodiments, one or more volatile salts can include, but are not limitedto, one or more of ammonium acetate, ammonium formate, ammoniumcarbonate, ammonium bicarbonate, triethylammonium acetate,triethylammonium formate, triethylammonium carbonate, trimethylamineacetate trimethylamine formate, trimethylamine carbonate, pyridinalacetate and pyridinal formate, or combinations thereof.

In other embodiments, formulations of use herein can include one or morenon-reducing disaccharides including, but not limited to, trehalose,sucrose and lactose, and additional glass forming agents, as necessary,including, but not limited to, hydroxyethyl starch, glycine, glycine andmannitol, cyclodextrin, and polyvinyl pyrrolidone (povidone) orcombinations thereof.

In certain aspects of the instant disclosure, immunogenic formulationsof broad-spectrum multi-targeted antigenic formulations, for example,can be lyophilized where the broad-spectrum construct remains intact. Inaddition, these combinations can reduce detrimental modifications tocritical neutralizing epitopes of an assembled multi-targeted complex.In other embodiments, broad-spectrum multi-targeted constructcompositions disclosed herein preserve antibody titer by increasingstability and/or decreasing disassembly or degradation. In certainembodiments, antigen compositions described herein can be stabilized toreduce antibody titer loss by lyophilization in buffers disclosedherein. In other embodiments, lyophilized multi-targeted antigeniccomplexes can be stored at elevated temperatures of about 40° C. toabout 50° C. to about 60° C. degrees for up to several weeks to severalmonths making it possible to store and transport these compositions atan increased temperature for a longer duration. In certain embodiments,immunogenic compositions of multi-targeted antigens or the like can befrozen on precooled shelves of a lyophilizer and dried under vacuumcreating an essentially dry powder formulation. In other embodiments,multi-targeted antigens or the like formulations including trehalose andammonium acetate can be lyophilized and dried to prolong shelf-life ofthe active agents for storage and transport or enhance immunogenicity.

“Multimeric” Protein

Generally, as the complexity of vaccine compositions increases, longterm stability decreases, especially at elevated temperatures. In somecases, vaccines compositions comprising multiple subunits (e.g.,multimeric) can have greater complexity than vaccine compositions thatare made of single proteins. For example, vaccine compositionscomprising antigens based on multiple capsomere subunits are generallymore complex and more resistant to forming stable vaccine compositions.In some cases, embedding a capsomere within glassy matrices formedduring lyophilization can enhance thermal stability of the vaccinecomposition by stabilizing the tertiary structure of the capsomeres.

In some embodiments, thermal stability of tertiary structure of a viralcomplex can be assessed by any method known in the art. In otherembodiments, thermal stability of tertiary structure of a viral complexcan be assessed using various methods including, but not limited to,front face fluorescence. For example, front face fluorescence can beused to examine tertiary structure of HPV 16 L1 capsomeres. In certainembodiments, front face fluorescence can use Acrylamide quenching toassess the tryptophan environment in each vaccine formulation, and aStern-Volmer constant can be calculated based on the fluorescence. Ahigh Stern-Volmer constant is generally indicative of greater tertiaryinstability, which allows tryptophan residues to be more easilyquenched. For example, a lower Stern-Volmer constant is generallyindicative of less tertiary instability (i.e., a more native proteinstructure), which reduces tryptophan quenching. Thus, these comparisonscan be made on a complex to assess stability of the complex at a giventemperature in compositions described herein.

In other embodiments, a non-reducing disaccharide disclosed herein caninclude one or more of trehalose, sucrose, lactose, or combinationsthereof. In some embodiments, the disaccharide concentration in aweight-to-volume (w/v) can be from about 1% to about 20%, or about 5% toabout 15% (w/v) in a liquid vaccine formulation prior to lyophilization.In other embodiments, the glass-forming agent can be trehalose presentin a concentration of from about 8% to about 20% w/v in the liquidvaccine formulation prior to lyophilization. In another embodiment, theglass-forming agent can be trehalose at a concentration of about 10% w/vin the liquid vaccine formulation or immunogenic composition prior tolyophilization.

In some embodiments, compositions disclosed herein can include ahypertonic buffer composed of volatile salts and a disaccharide agent atvarious concentrations (1.0% to 20% (w/v)) prior to lyophilization. Inother embodiments, a broad-spectrum multi-targeted antigenic constructdisclosed herein included in a stabilizing composition forlyophilization or other purpose can be from about 0.01 mg/mL to about5.0 mg/mL, or about 0.01 mg/mL to about 3.0 mg/mL; or about 0.01 mg/mLto about 2.0 mg/mL; or about 0.05 mg/mL to about 1.5 mg/mL. In someembodiments, a multi-targeted antigen construct can be formulated in astabilizing composition for lyophilization or other purpose can be fromabout 0.05 mg/mL to about 2.0 mg/mL.

In some embodiments, stability of vaccine or immunogenic compositionsdisclosed herein can be enhanced by the addition of nonionicsurfactants. In accordance with these embodiments, surfactants can beadded to vaccine or immunogenic formulations at concentrations rangingfrom approximately 0.1 times the critical micelle concentration of thesurfactant in the vaccine composition, to approximately 20 times thecritical micelle concentration of the surfactant in the vaccinecomposition before, during or after lyophilization of the composition.Suitable nonionic surfactants include, but are not limited to,polysorbates such as Tween 20, Tween 40, Tween 60 and Tween 80,poloxamers for example Poloxamer 188 and Polaxamer 407, Poloxamer 235,Poloxamer 335, Brij, alkylphenol hydroxypolyethylene surfactants such asTriton X100, Triton X114 and Triton X405, and oligoethylene glycolmonoalkyl ethers such as Genapol.

In some embodiments, thermal stability of tertiary structure of abroad-spectrum multi-targeted complex can be assessed by any methodknown in the art. In other embodiments, thermal stability of tertiarystructure of broad-spectrum multi-targeted complexes can be assessedusing various methods including, but not limited to, front facefluorescence. For example, front face fluorescence can be used toexamine tertiary structures of certain complexes. In certainembodiments, front face fluorescence can use acrylamide quenching toassess the tryptophan environment in each vaccine formulation, and aStern-Volmer constant can be calculated based on the fluorescence. Ahigh Stern-Volmer constant is generally indicative of greater tertiaryinstability, which allows tryptophan residues to be more easilyquenched. For example, a lower Stern-Volmer constant is generallyindicative of less tertiary instability (i.e., a more native proteinstructure), which reduces tryptophan quenching. Thus, these comparisonscan be made on a complex to assess stability of the complex at a giventemperature in compositions described herein.

VLPs and Capsomeres

Virus-like particles or VLPs: the capsid-like structures that resultupon expression and assembly of a papillomavirus L 1 DNA sequence aloneor in combination with an L2 DNA sequence. VLPs are morphologically andantigenically similar to authentic virions. VLPs may be produced invivo, in suitable host cells or may form spontaneously upon purificationof recombinant L1 and/or L2 proteins. Additionally, they may be producedusing capsid proteins L1 and L2, fragments or mutated forms thereof,e.g., L1 or L2 proteins that have been modified by the addition,substitution or deletion of one or more amino acids. L1 and L2 mutantsthat fall within the scope of the present invention are those that uponexpression present at least one native PV conformational epitope.Methods to assemble VLPs are known in the art, as would be readilyappreciated and is understood by one of ordinary skilled based on thepresent disclosure.

Correctly-folded L1 or L2 protein: L1 or L2 protein, fragment thereof,or mutated form thereof, (either monomeric, in the form of smalloligomers (dimers-tetramers) or capsomeres), which, upon expression,assumes a conformational structure that presents one or moreconformational HPV L1 or L2 epitopes present on native viral capsids orVLPs and is suitable for assembly into VLPs. In the present invention, acorrectly folded HPV L1 or L2 protein will present one or more HPV L1 orL2 conformational epitopes.

A conformational LI or L2 HPV epitope: generally refers to an epitopeexpressed on the surface of correctly-folded L1 or L2 protein which isalso expressed by an L1 or L2 protein or fragment, or mutated formthereof, which is also expressed by an L1 or L2 protein of acorresponding wild-type, infectious HPV. It is well accepted by thoseskilled in the art that the presentation of conformational epitopes isessential to the efficacy (both as prophylactic and diagnostic agents)of HPV L I or L2 protein immunogens.

A conformational neutralizing L1 or L2 HPV epitope: generally refers toan epitope expressed on the surface of correctly-folded L1 protein,fragment or mutated form thereof, which is also expressed by an L1 or L2protein of a corresponding wild-type, infectious HPV, and which elicitsneutralizing antibodies. It is well accepted by those skilled in the artthat the presentation of conformational neutralizing epitopes isessential to the efficacy (both as prophylactic and diagnostic agents)of HPV L1 or L2 protein immunogens.

Embodiments herein provide for compositions and methods for stabilizingvaccine or immunogenic formulations and prolong stability during storagefor HPV vaccines or immunogenic compositions. In some embodiments, anHPV chimeric protein of compositions disclosed herein can include apapillomavirus L2 capsid polypeptide having a papillomavirus capsidprotein L1-binding domain and a second polypeptide comprising at leastone immunogenic epitope, wherein the polypeptides are fused at theiramino or carboxy terminal ends. The papillomavirus L2 capsid polypeptidecan include the full-length papillomavirus L2 capsid protein as well astruncated versions of the L2 protein containing an L1 capsid proteinbinding region. Additionally or alternatively, the present disclosureprovides a chimeric protein comprising a papillomavirus L1 proteinlinked by at least one amino acid to a second polypeptide comprising atleast one immunogenic epitope. The papillomavirus L1 capsid polypeptidecan include the full-length papillomavirus L1 capsid protein as well astruncated versions of the L1 protein.

Certain embodiments can include vaccine formulations of capsomeres,including but not limited to, truncated L1 with or without L2 viralproteins. In some embodiments, capsomeres include truncated L 1proteins. Truncated proteins contemplated herein can include thosehaving one or more amino acid residues deleted from the carboxy terminusof the L1 protein, or one or more amino acid residues deleted from theamino terminus of the L 1 protein, or one or more amino acid residuesdeleted from an internal region of the protein. In accordance with theseembodiments, a capsomere vaccine formulation or immunogenic compositioncan include L1 proteins truncated at the carboxy terminus.

Immunogenic epitopes are those that confer protective immunity, allowinga mammal or other animal to resist (delayed onset of symptoms or reducedseverity of symptoms), as the result of its exposure to the antigen of apathogen, disease or death that otherwise follows contact with thepathogen. Protective immunity can be achieved by one or more of thefollowing mechanisms: mucosal, humoral, or cellular immunity. Mucosalimmunity is primarily the result of secretory IgA (sIGA) antibodies onmucosal surfaces of the respiratory, gastrointestinal, and genitourinarytracts. The sIGA antibodies are generated after a series of eventsmediated by antigen-processing cells, B and T lymphocytes that result insIGA production by B lymphocytes on mucosa-lined tissues of the body.“Humoral immunity” is the result of IgG antibodies and IgM antibodies inserum. “Cellular immunity” can be achieved through cytotoxic Tlymphocytes or through delayed-type hypersensitivity that involvesmacrophages and T lymphocytes, as well as other mechanisms involving Tcells without a requirement for antibodies. The primary result ofprotective immunity is the destruction of the pathogen or inhibition ofits ability to replicate itself.

Embodiments of the present disclosure can include a complex includingchimeric proteins and further include a papillomavirus L1 polypeptide,protein or fragment thereof, or substantially identical protein orfragments. Papillomavirus L1 polypeptides of the present inventioninclude polypeptides that retain their ability to bind to papillomavirusL2 polypeptides of the present invention. The complexes disclosed hereincan include L1 capsid protein fragments that upon expression presentconformational, neutralizing epitopes. These fragments can include fulllength papillomavirus L1 capsid proteins as well as internal, carboxy-and amino-terminal deletions, and proteins having specific cysteinemutations that prevent assembly into VLPs. The deletion may range insize from 1 to about 100 amino acids, preferably 1 to 50 amino acids,and more preferably from about 1 to 25 amino acids. It is essential thatthe deletion still allow for the expression of a capsid protein, e.g.,HPV L1 protein, that when expressed in fused or non-fused form presentsat least one conformational, neutralizing epitope.

Complexes disclosed herein can be in the form of a capsomere. Capsomeresof the present invention will generally have a stoichiometry of aboutone chimeric protein of the present invention to about fivepapillomavirus L1 capsid proteins, although capsomeres of greater orlesser stoichiometry are also contemplated.

In another embodiment, the capsomeres of the present invention can beassembled into a VLP. In this embodiment, assembly can be performedusing methods known in the art. The present invention includes methodsto assemble a VLP using capsomeres of the present invention at acidic tophysiological pH. Most preferred are methods to assemble VLPs usingcapsomeres of the present invention at physiologic pH. In the case ofpolypeptide sequences which are less than 100% identical to a referencesequence, the non-identical positions are preferably, but notnecessarily, conservative substitutions for the reference sequence.

Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine. Similarminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art.

Viral proteins of the present disclosure may be derived from anypapillomaviruses, including human papillomavirus. For example, HPV L1and L2 DNA sequences exhibit significant homology to L 1 s and L2s ofdifferent serotypes of HPV. Therefore, HPV L1 or L2 nucleic acidsequences can be obtained, as would be understood by one of ordinaryskill in the art based on the present disclosure.

In some embodiments, the HPV L1 or L2 DNA disclosed herein derived froman HPV which is involved in cancer or condylomata acuminata, e.g.,HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52,and HPV-56 are involved in cancer, and HPV-6, HPV-11, HPV-30, HPV-42,HPV-43, HPV44, HPV-54, HPV-55, and HPV-70, are involved in warts.However, the subject capsid proteins may be produced using any HPV L1DNA.

Proteins and capsomeres disclosed herein can be produced in a variety ofways, including production and/or recovery of natural proteins,production and/or recovery of recombinant proteins, and/or chemicalsynthesis of the proteins. The proteins and polypeptides disclosedherein can be expressed in a prokaryotic microbial host, e.g., bacteriasuch as E. coli, that can be cultured under conditions that favor theproduction of capsid proteins. This will largely depend upon theselected host system and regulatory sequences contained in the vector,e.g., whether expression of the capsid protein requires induction.Proteins and polypeptides of the present disclosure may also beexpressed in any host cell that provides for the expression ofrecoverable yields of the polypeptides in appropriate conformation.Suitable host systems for expression of recombinant proteins are wellknown and include, by way of example, bacteria, mammalian cells, yeast,and insect cells. One expression system of use to produce complexesdisclosed herein can include E. coli expression system used in theExamples, as this system provides for high capsomere yields. However,HPV L1 and L2 proteins, as well as other viral capsid proteins, can beproduced in other systems. For example, yeast and baculovirus-infectedinsect cell cultures can be used.

Suitable vectors for cloning and expressing polypeptides of the presentinvention are well known in the art and commercially available. Further,suitable regulatory sequences for achieving cloning and expression,e.g., promoters, polyadenylation sequences, enhancers and selectablemarkers are also well known. The selection of appropriate sequences forobtaining recoverable protein yields is routine to one skilled in theart.

Other embodiments can include polynucleotides that encode chimericproteins and complexes/capsomeres. Accordingly, any nucleic acidsequence, which encodes the amino acid sequence of chimeric proteins andcomplexes/capsomeres, can be used to generate recombinant molecules thatexpress chimeric proteins and complexes/capsomeres. It will beappreciated by those skilled in the art based on the present disclosurethat as a result of the degeneracy of the genetic code, a multitude ofnucleotide sequences encoding chimeric proteins and complexes/capsomeresof the present disclosure, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. Thus, the disclosure contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring chimeric proteins andcomplexes/capsomeres of the present disclosure, and all such variationsare to be considered as being disclosed.

Chimeric proteins and capsomeres have application in both prophylacticand therapeutic vaccines and diagnostics. The suitability of thechimeric proteins and capsomeres produced for use as vaccines or asdiagnostic agents can be confirmed by reaction with antibodies ormonoclonal antibodies which react or recognize conformational epitopespresent on the intact vision and based on their ability to elicit theproduction of neutralizing antiserum. Suitable assays for determiningwhether neutralizing antibodies are produced are known to those skilledin the art based on the present disclosure. This is an essentialcharacteristic of HPV capsid proteins or other viral capsid proteins,which are to be used in HPV or other viral vaccines. In this manner, itcan be verified whether the polypeptides of the present disclosure willelicit the production of anti-HPV neutralizing antibodies. Thus, otherexpression vectors and expression systems can be tested for use in thepresent disclosure.

Certain embodiments disclosed herein concern using adjuvants to increaseimmunogenicity of viral complex compositions or formulations forvaccines. Adjuvants are typically substances that generally enhance theimmune response of a patient to a specific antigen. Suitable adjuvantsinclude, but are not limited to, other bacterial cell wall components,aluminum based salts, calcium based salts, silica, polynucleotides,toxins, such as cholera toxin, toxoids, such as cholera toxoid, serumproteins, other viral coat proteins, other bacterial-derivedpreparations, block copolymer adjuvants, such as Hunter's TITERMAXadjuvant (VAXCEL, Inc., Norcross, Ga.); RIBI adjuvants (available fromRibi ImmunoChem Research, Inc., Hamilton, Mont.) and saponins and theirderivatives, such as QUIL A (available from Superfos Biosector A/S,Denmark). Carriers are typically compounds that increase half-life of acomposition or agent in a subject. Suitable carriers include, but arenot limited to, polymeric controlled release formulations, biodegradableimplants, liposomes, bacteria, viruses, oils, esters and glycols.

Certain embodiments of the present application include polypeptides thatelicit an immune response to an HPV antigen in a subject. An elicitedimmune response may be either prophylactic, preventing later infectionby the specific viral type targeted, or may be therapeutic, reducing theseverity of disease. An immune response includes a humoral, e.g.,antibody, response to that antigen and/or a cell-mediated response tothat antigen. Methods to measure an immune response are known to thoseskilled in the art. If one or both types of immune response are present,they may protect a subject from any disease caused by an agent, forexample, by the agent from which the viral complex was derived. Inaccordance with the present disclosure, the ability of an immunogeniccomposition to protect or treat a subject in need thereof from diseasecan refer to the ability of a capsomere or chimeric protein of thepresent disclosure to treat, ameliorate and/or prevent disease orinfection caused by the agent or cross reactive agent, by eliciting animmune response against an antigen derived from the disease-causingagent and contained within a protein or capsomere of the presentdisclosure. It is to be noted that a subject may be protected by animmunogenic composition disclosed herein even without detection of ahumoral or cell-mediated response to the immunogenic composition.Protection or reducing the risk of developing a viral infection can bemeasured by methods known to those skilled in the art.

In certain aspects, because it is known that more than one HPV type maybe associated with an HPV infection, vaccines or immunogeniccompositions can include stable HPV capsid proteins derived from morethan one type of HPV where the compositions have been lyophilized withglass-forming excipients to increase their stability to non-refrigeratedtemperatures. For example, HPV 16 and 18 are known to be associated withcervical carcinomas, therefore, a vaccine for cervical neoplasia caninclude VLPs of HPV 16; of HPV 18; or both HPV 16 and 18. In fact, avariety of neoplasias are known to be associated with PV infections. Forexample, HPVs 3a and 10 have been associated with flat warts. A numberof HPV types have been reported to be associated with epidermodysplasiaverruciformis (EV) including HPVs 3a, 5, 8, 9, 10, and 12. HPVs 1, 2, 4,and 7 have been reported to be associated with cutaneous warts and HPVs6b, 1 1 a, 13, and 16 are associated with lesions of the mucusmembranes. Thus, the subject vaccine formulations may comprise a mixtureof capsid proteins or fragments derived from different BPV typesdepending upon the desired protection.

Other embodiments concern pharmaceutical immunogenic compositions foruse in reducing the risk of onset or treating a condition caused by apathogenic virus (e.g., HPV). Any known pharmaceutically acceptableexcipient is contemplated herein.

Yet another aspect of the present disclosure is a method to elicit animmune response to a chimeric protein or capsomere of a lyophilized ordehydrated composition (after hydration), comprising administering tothe subject a composition disclosed herein. The vaccines will beadministered in prophylactically or therapeutically effective amounts.That is, in amounts sufficient to produce a protective immunologicalresponse. Generally, the vaccines will be administered in dosagesranging from about 0.1 mg protein to about 20 mg protein, more generallyabout 0.001 mg to about 1 mg protein. Single or multiple dosages can beadministered.

Administration of the subject capsid protein-containing vaccines may beeffected by any pharmaceutically acceptable means, e.g., parenterally,locally or systemically, including by way of example, oral, intranasal,intravenous, intramuscular, and topical administration. The manner ofadministration is affected by factors including the natural route ofinfection. The dosage administered will depend upon factors includingthe age, health, weight, kind of concurrent treatment, if any, andnature and type of the particular viral, e.g., human, papillomavirus.The vaccine may be employed in dosage form such as capsules, liquidsolutions, suspensions, or elixirs, for oral administration, or sterileliquid formulations such as solutions or suspensions for parenteral orintranasal use.

In yet other embodiments, multi-targeted antigen complexes can belyophilized and stored in elevated temperatures of about 40° C. to about60° C. for a pre-determined period of days to months (e.g. 1 day, 1week, several weeks to a month or more) to enhance immunity whenintroduced to a subject to a broad range of types or serotypes ofpathogenic organisms. For example, enhancing epitope availability orenhancing neutralization effects of a composition as a result ofexposure to these elevated temperatures during storage. In certainembodiments, enhanced immunogenicity can occur in simultaneously to therepresented antigens of the complex. This aspect of the instantinvention is surprising and unexpected as elevated temperaturestypically have an adverse effect on immunogenicity of multi-complexedagents. In accordance with these embodiments, exposure to increasedtemperatures as reference above of a stabilized, lyophilizedmulti-targeted antigen (e.g. RG1 HPV VLP), of the instant application,can increase cross-reactivity of the reconstituted complex againstmultiple pathogenic types or serotypes when introduced to a subject. Incertain embodiments, a subject contemplated herein can be a humansubject or other mammalian subject such as a pet or livestock.

Certain embodiments disclosed herein can include kits of use for storageand transport of one or more multi-targeted antigen construct disclosedherein, one or more container and/or one or more lyophilizedmulti-targeted antigen construct. In accordance with these embodiments,a kit can include a container having a lyophilized multi-targetedantigen construct in trehalose and ammonium acetate or similar agent asdisclosed herein.

EXAMPLES

This disclosure is further illustrated by the following non-limitingexamples. All scientific and technical terms have the meanings asunderstood by one with ordinary skill in the art. The examples whichfollow illustrate the methods in which the chimeric compositions of thepresent disclosure may be prepared and used and are not to be construedas limiting the disclosure in sphere or scope. The methods may beadapted to variation in order to produce compositions embraced by thisdisclosure but not specifically disclosed. Further, variations of themethods to produce the same compositions in somewhat different fashionwill be evident to one skilled in the art based on the presentdisclosure.

Example 1

In certain exemplary methods, it is known that liquid vaccines thatcontain microparticulate adjuvants such as aluminum hydroxide may beparticularly prone to damage resulting from accidental freezing, becauseof the tendency of these adjuvants to agglomerate during freezing.Limitations of refrigerated storage for vaccines are even morepronounced when delivering vaccines to a developing country or region.Lyophilization can be used to embed vaccine antigens and adjuvantswithin glassy organic matrices, providing an environment wherecombination of low molecular mobility and low moisture content assist inminimizing antigen degradation. By utilizing high concentrations ofglass-forming excipients and in certain cases rapid freezing rates,agglomeration and ultimate degradation caused by microparticulateadjuvants can be avoided or minimized during the lyophilization process.

Embodiments of the present disclosure can be used to increase stabilityand/or immunogenicity of vaccine formulations through the use oflyophilization to preserve or stabilize the immunogenic complexes.Lyophilization of various vaccine formulations have been demonstrated todecrease protein degradation by, for example, immobilizing vaccinecomponents in a high viscosity glassy matrix with low water content. Insome cases, a high glass transition temperature allows for storage in aglassy state at elevated temperatures without significantly increasingprotein degradation. For example, trehalose can be used to stabilize theprotein in both the liquid and the solid state and can increase theglass transition temperature. Storage of the vaccine formulations belowthe glass transition temperature allows for the formulation to be storedin a glassy state.

Lyophilized formulations of the present disclosure generally have lowwater content and do not absorb water during storage. Low water contentcan help prevent degradations from occurring. Although vaccine particlesizes can vary, it was found that cooling rate and trehaloseconcentration are two factors that can affect aluminum adjuvant particlesize after lyophilization. However, particle size was found to remainconstant after storage and antigen tertiary structure was found to bepreserved after lyophilization.

In some embodiments, the immunogenicity of vaccine formulations thathave undergone lyophilization can be increased by the addition ofadjuvants. For example, aluminum salts such as aluminum hydroxide, cancreate a humoral (Th2) response, and Toll-like receptor 4 (TLR4)agonists such as glycopyranoside lipid A (GLA), can create a cellular(Th1) response. The addition of agonists such as these can increaseantibody titers and increase the rate of seroconversion, even afterstorage at 40° C.

Vaccine Characterization

In certain exemplary methods, it is desirable to store vaccineformulations below both the protein melting temperature and thelyophilized formulation glass transition temperature. For example, theonset melting temperature of the HPV 16 L1 capsomere was determined tobe approximately 60° C. (not shown). The melting temperature of HPV VLPstypes 6, 11, 16, and 18 found in GARDISIL are all above this temperature(Shank-Retzlaff 2006). The onset glass transition temperature forlyophilized placebo formulations was found to be 97.2° C.±3.4° C., andwhen an adjuvant was added (e.g., aluminum salt), the onset glasstransition temperature for lyophilized placebo formulations was found tobe between 102.6° C.±5.2° C. The addition of protein to theseformulations did not significantly affect the glass transitiontemperature. By storing vaccines below both the protein meltingtemperature and glass transition temperature, protein will notimmediately denature upon storage and the lyophilized vaccines will bestored in a glassy state with extremely low mobility. A storagetemperature of 50° C. was chosen to evaluate stability for subsequentexperimental evaluation.

Certain exemplary embodiments of the vaccines or immunogeniccompositions of the present disclosure were characterized in liquid formbefore lyophilization, immediately after lyophilization reconstitution,and after storage at 50° C. for 12 weeks in both liquid and lyophilizedforms. Vaccines were analyzed for capsomere appearance, for example,front face fluorescence was used for tertiary structure, V5 and L1assays were used for conformational epitope reactivity, and FlowCAM wasused for particle size and concentration.

As illustrated in FIGS. 1A-1C, transmission electron microscopy (TEM)was used to visualize HPV 16 L1 capsomeres before lyophilization (A),immediately after lyophilization and reconstitution (B), and afterstorage in the lyophilized and reconstituted state (C). Beforelyophilization, HPV 16 capsomeres are uniformly spherical in nature.After lyophilization and reconstitution, capsomere proteins are similarto their initial state. Additionally, storing the lyophilized vaccinefor 12 weeks at 50° C. did not affect capsomere appearance. These datademonstrate that the quaternary structure of HPV 16 L1 capsomeres ispreserved after lyophilization. The scale bar represents 100 nm.

Additionally, capsomeres were maintained as a pentamer of L1 proteinsduring lyophilization as demonstrated by retention of the capsomere peakin size exclusion chromatography. The area under the peak was integratedto be 422, 0, 413, and 415 arbitrary units for liquid HPV 16 L1capsomere, stored liquid HPV 16 L1 capsomere, lyophilized HPV 16 L1capsomere, and stored lyophilized HPV 16 L1 capsomere respectively (datanot shown). After storage at 50° C. in the liquid state the capsomereprotein was completely lost, demonstrating that the instant compositionsand methods were capable of preserving/stabilizing the complex asobserved by presence of a capsomere peak in the treated conditions.

Example 2

In another exemplary method, to examine the tertiary structure of HPV 16L1 capsomere proteins, front face fluorescence was used. In one example,the tryptophan environment in each vaccine formulation was assessed,acrylamide quenching was performed, and a Stern-Volmer constant wascalculated. A high Stern-Volmer constant is indicative of more unfoldingof the protein allowing for tryptophan residues to be more easilyquenched, whereas a lower Stern-Volmer constant indicates that thetryptophan residues were more difficult to access, thus indicating amore native-like protein tertiary structure. The Stern-Volmer constantremained constant for the initial liquid state, the reconstituted andlyophilized state, and for the lyophilized incubated and reconstitutedstate (e.g., after storage), for both protein and protein+alum vaccines,as illustrated in FIG. 2. These data demonstrate that the tertiarystructures of the vaccines in these embodiments were retained afterlyophilization and storage. The protein+alum vaccines had a slightlylower Stern-Volmer constant which may be due to tryptophan residuesadsorbing the aluminum hydroxide adjuvant and therefore being lessaccessible to acrylamide.

Experiments were also conducted to demonstrate the reactivity of HPV 16L1 capsomeres to two antibodies, V5 (FIG. 3A) and L1 (FIG. 3B). L1antibody reactivity was used to monitor the structure of many epitopesof the L1 subunit in the capsomere, and V5 antibody reactivity was usedto monitor a conformational neutralizing epitope presented by thepentamer. As demonstrated, reactions with both antibodies were retainedduring the lyophilization process, as well as after elevated temperaturestorage in the lyophilized state (FIG. 4). The positive control used forcomparison was a fresh sample of the HPV 16 L1 capsomere protein, whilethe negative control used was a polyomavirus structural protein, VP1, astructural equivalent to L1.

Example 3 Vaccine Immunogenicity

Because HPV 16 L1 capsomere protein was preserved during storage asprovided above, immunogenicity of the stored vaccine as compared to theinitial vaccine was evaluated. Particle concentrations were assessedprior to testing immunogenicity. As shown in FIG. 4, the concentrationsof particles greater than 2 microns (pm) remained fairly constantthrough lyophilization and storage, with approximately 5×104particles/mL for placebo groups and protein formulations and 5×106particles/mL for placebo+alum and protein+alum formulations.

Vaccine immunogenicity was assessed by measuring total anti-HPV 16 L1capsomere antibody titers (FIG. 5A) as well as neutralizing antibodytiters (FIG. 5B). A dose response relationship was demonstrated forlyophilized vaccines (protein (P) and protein+alum (PA)), at doses of 7,5, 3, and 1 μg/dose, for GARDASIL at doses of 5, 3, and 1 μg/dose, andfor CERVARIX at doses of 4, 3, 2, and 1 μg/dose. All of the dosesadministered were in the linear range based on the murine model used.All doses of formulations containing the adjuvant aluminum hydroxide hadsignificantly (p<0.05) greater immune responses than formulationscontaining only protein after one and two injections, except the 5 p.gdose after two injections (p=0.46). The addition of aluminum hydroxideincreased the antibody titers one order of magnitude from protein alone.GLA did not significantly increase the antibody titers (p>0.05) afterone or two injections. Additionally, lyophilized vaccines containingadjuvants preformed equally as well if not better than commerciallyavailable vaccines based on total IgG antibody titers.

Lyophilized vaccine formulations were incubated at 50° C. for 12 weeksand then injected into mice at 5 and 1 μg/dose since these were found tobe in the linear range of the immune response. GARDASIL and CERVARIXwere injected at 5 and 1, and 4 and 1 μg/dose, respectively. Due to alimited supply of GARDASIL, only one dose was administered for theincubated vaccines. As illustrated in FIG. 6A, lyophilized vaccinesproduced anti-HPV 16 L1 capsomere antibody titers similar to theirnon-incubated counterparts with the exception of the protein onlyvaccines at a 5 [tg dose after two vaccine injections. Neutralizingantibody titers are illustrated in FIG. 6B. GARDASIL had similar titervalues after one injection, but CERVARIX had significantly (p=0.008)decreased titers. The predicted half-life of GARDASIL at 42° C. is a fewmonths; however, these data demonstrate that at a longer incubationtime, even at 50° C., high antibody titers were maintained.

FIGS. 7A and 7B illustrate graphical representations of dose responsecurves for an antibody neutralization study (A) and an antibodyincubation study in mice (B) for various vaccine formulations, accordingto embodiments of the present disclosure.

Taken together, these data demonstrate that lyophilized HPV 16 L1capsomere vaccines remained stable and highly immunogenic after anelevated storage temperature of about 50° C. for the 12 weeks tested,stabilizing the formulation for storage, delivery and use. Thepotentially lower cost of the capsomere protein, in conjunction with thehigh thermostability of the lyophilized vaccine, makes thesepreparations excellent candidates for HPV vaccines, for example, fordeveloping countries where access to these types of vaccines is reduced.

Example 4

In another exemplary method, preparation of HPV vaccine formulations,alum-adjuvanted HPV vaccine formulations and alum- and MPLA-adjuvantedHPV vaccine formulations containing capsomeres of HPV16 LI, HPV18 L1,HPV31 L1 or HPV45 L1, as well as tetravalent HPV vaccine formulationscontaining mixtures of capsomeres of HPV16 L1, and alum- andMPLA-adjuvanted HPV vaccine formulations containing capsomeres of HPV16LI, HPV18 L1, HPV31 L1 or HPV45 L1 were generated.

In certain examples, aqueous protein solutions were formulated tocontain either HPV 16, 18, 31, or 45 capsomeres at a concentration of0.05 mg/mL. Formulations were prepared in 100 mM histidine buffer at pH7.1 with 9.5 w/v % trehalose as 1 mL aliquots. a, a-Trehalose dehydrateand L-histidine monohydrochloride monohydrate were purchased fromSigma-Aldrich (St. Louis, Mo.). Each HPV strain was formulated in threeways: (i.) with no adjuvant present, (ii.) with 0.5 mg/mL aluminum fromALHYDROGEL and (iii.) with 0.5 mg/mL aluminum from ALHYDROGEL with 0.05mg/mL MPLA. ALHYDROGEL adjuvant 2% (also referred to herein as alum)(e.g., E.M. Sergeant Pulp & Chemical Co, Inc., Clifton, N.J.). Syntheticmonophosphoryl lipid A (MPLA) a glyclopyranoside lipid An adjuvant;Avanti Polar Lipids, Inc. Alabaster, Ala.

In one example, formulations containing ALHYDROGEL were rotatedend-over-end at 8 rpm in 1.5 mL polypropylene microcentrifuge tubes at4° C. for 1 hour to allow capsomere adsorption onto adjuvant.Additionally, a formulation containing 0.0125 mg/mL of all four HPVcapsomere types (16, 18, 31, and 45) was made without adjuvant as acontrol.

Comparisons of TEM images that were recorded before lyophilization andafter lyophilization and reconstitution (FIGS. 8-11) for formulationscontaining capsomeres of each type alone (HPV16 LI, HPV18 L1, HPV31 L1or HPV45 L1), as well as tetravalent vaccine formulations containing allfour HPV capsomere types (FIG. 12), demonstrated that the pentamericconformation of the HPV capsomere were retained through thelyophilization and reconstitution process.

Western blot analysis of aluminum hydroxide-adjuvanted formulations ofvaccines containing HPV16 L1 capsomeres, HPV18 L1 capsomeres, HPV31 L1capsomeres, or HPV45 L1 capsomeres sampled prior to lyophilization andafter lyophilization and reconstitution demonstrated that antigenicepitopes were retained after lyophilization and reconstitution (FIGS.13A-13C). Furthermore, samples from tetravalent vaccine formulationscontaining aluminum hydroxide adjuvant also showed retention ofantigenic epitopes after lyophilization and subsequent reconstitution,as measured using ELISA assays.

In FIGS. 8A and 8B, TEM images of HPV16 L1 capsomeres were capturedbefore lyophilization (A) and after lyophilization and reconstitution(B).

In FIGS. 9A and 9B, TEM images of HPV18 L1 capsomeres were capturedbefore lyophilization (A) and after lyophilization and reconstitution(B).

In FIGS. 10A and 10B, TEM images of HPV31 L1 capsomeres were capturedbefore lyophilization (A) and after lyophilization and reconstitution(B).

In FIGS. 11A and 11B, TEM images of HPV45 L1 capsomeres were capturedbefore lyophilization (A) and after lyophilization and reconstitution(B).

In FIG. 12, TEM images of a tetravalent vaccine formulation containingHPV16 L 1, HPV18 L1, HPV31 L1, and HPV45 L1 capsomeres were capturedafter lyophilization and reconstitution. These data demonstrate that allof the above HPV vaccine formulations exhibited the pentamericconformation of the HPV capsomere throughout the lyophilization andreconstitution process.

As illustrated in FIGS. 13A and 13B, vaccine formulations containingHPV16 L1, HPV18 L1, HPV31 L1, or HPV45 L1 capsomeres were subjected toSDS Page and Western Blot analysis before lyophilization (A), and afterlyophilization and reconstitution (B). Additionally, as illustrated inFIG. 13C, a tetravalent formulation comprising HPV16 L1, HPV18 L1, HPV31L1, and HPV45 L1 capsomeres of HPV16 L1, HPV18 L1, HPV31 L1, and HPV45L1 capsomeres was also subjected to SDS Page and Western Blot analysisbefore lyophilization (“PRE”) and after lyophilization andreconstitution (“POST”). For Western Blot analysis, a protein ladder wasincluded in the lane directly to the left of each of the vaccineformulation samples. These data demonstrate that the pentamericconformation of each of the above HPV vaccine formulations wasconservation the throughout lyophilization and reconstitution process.

Example 5

In certain exemplary methods, broad-spectrum HPV immunogeniccompositions were tested in various formulations for stability atelevated temperatures. In one method, an ELISA assay was performed toassess titer of various formulations subjected to lyophilization andstorage for prolonged stability. In exemplary FIG. 14, immune serasamples raised against lyophilized and reconstituted formulations of anexemplary construct, RG1-VLP that had been stored under varioustemperature conditions were tested by an HPV16 L1-VLP and RG1 peptideELISA in 4-fold serial dilutions (1:200-1:204,800). Rabbit sera raisedagainst HPV16 L1-VLP and RG1-VLP, and a BPV L1-raised monoclonalantibody, were used as positive or negative controls. Titers were gradedpositive for mean OD values greater than OD of pre-sera+3 standarddeviations. n.d. indicate not determined. See FIG. 14 where stability isdemonstrated at various temperatures up to an elevated temperature ofabout 50° C.

As illustrated in FIG. 14, titers were maintained at all temperaturestested.

Example 6

In another exemplary method, a pseudovirion-based neutralization assay(PBNA) was performed after storage of the HPV constructs at varioustemperatures and times. As illustrated in FIG. 15A and FIG. 15B L 1-PBNA(See for example, Buck 2004, 2005) was performed to detect neutralizingantibodies against hr. HPV16, and cross-neutralizing antibodies againsthr. HPV18, 31, 39 and cutaneous Beta type HPV5. L2-PBNA (See for exampleDay 2012) was performed against HPV39 and HPV5 to more sensitivelydetect potential cross-neutralization, and improved antibody titersdetected were demonstrated (See the bold print). Surprisingly,cross-neutralizing titers against multiple HPV types such as HPV types8, 18 31 and 39 were enhanced (instead of reduced) after a thermaltreatment consisting of incubation for 1 month at 50° C.

Example 7

In another exemplary method, splenocytes were harvested from groupsimmunized with lyophilized RG1-VLP in certain exemplary compositionsstored for 1 month at 4° C., 20° C., 37° C. or 50° C. and ex vivostimulated with either HPV16 or HPV18 L 1 -VLP, or medium andStaphylococcus aureus enterotoxin A (SEA) as controls. Evaluation wasperformed using an ImmunoSpot® Analyzer (CTL) and Immunospot Software5.0. (See for example, FIG. 16)

It was demonstrated in these exemplary methods that high-titerantibodies directed against HPV16 L1-VLP and the RG1 peptide weredetected in all lyophilized RG1-VLP-raised immune sera by ELISA (FIG.14). Notably, antibody titers were maintained even when lyophilizedRG1-VLP were stored at elevated temperatures. Immune sera wereneutralizing by L1-PBNA against HPV16 (titers of 3,200-51,200) andcross-neutralizing against hr HPV18, 31 and 39, and Beta HPVS (titersranging from 50-3,200) in the majority of temperature groups (FIGS. 15Aand 15B). Improved cross-neutralization was detected by more sensitiveL2-PBNA particularly against HPV39 (titers of 50-800) and for somegroups against HPVS (titers of <50-200). In this example, followingincubation at higher temperatures lyophilized RG1-VLP maintain theability to induce (cross-)neutralization with a trend towards reducedcross-neutralization seen in the highest temperature group (50° C.). ByELISPOT (FIG. 16), IFNy was induced by stimulation of splenocytes withHPV16 L 1 -VLP, but not HPV18 L1-VLP, in all tested storage temperaturegroups, which indicates maintained ability to raise a T cell responseregardless of storage temperature of the RG1-VLP.

Example 8 Preparation of Lyophilized RG1-VLPs in Thermostable GlassyMatrices

In one exemplary method, RG1-HPV VLPs were buffer-exchanged into asolution containing 100 mM histidine, pH7.1 Scanning electronmicrographs (See FIG. 17) of the RG1-HPV VLP solutions revealed thepresence of intact virus-like particles, with spiky protuberances. Thesolutions of RG1 HPV16 VLPs were mixed with trehalose and alum to form amixture containing 10 wt/vol % trehalose, 0.5 mg/mL Alhydrogel® alummicroparticles, and 0.1 mg/mL RG1 HPV VLPs. Other solutions wereprepared in a similar fashion, but additionally contained 0.05 mg/mL ofthe immune co-stimulatory agent monophosphoryl lipid A. 1 mL aliquots ofthe solutions were filled into 3 mL Schott Fiolax lyophilization vials.The vials were placed on precooled (−40° C.) shelves of a Lyostar pilot-scale lyophilizer. Samples were dried under vacuum (60 mTorr) and vialswere sealed under nitrogen. Samples of the lyophilized formulations werestored in temperature-controlled incubators at 4, 20, 37 and 50 ° C. fora period of 1 day, 1 week, and 1 month. After storage, samples werereconstituted with 1 mL of water for injection, and 100 microliter dosesof the resulting solution were administered to mice. FIG. 4 representsan exemplary image of a scanning electron micrograph of RG1-HPV VLPsdemonstrating intact virus-like particles after buffer exchange into 100mM histidine, pH 7.1

Materials and Methods

High purity a,a-trehalose dihydrate and sulfuric acid were purchasedfrom Mallinckrodt Baker (Phillipsburg, N.J.). Histidine HCl,triethanolamine, and bovine serum albumin (BSA) were purchased fromSigma-Aldrich (St. Louis, Mo.). Two percent ALHYDROGEL (aluminumhydroxide adjuvant) was obtained from Accurate Chemicals and ScientificCorp (Westbury, N.Y.). Lyophilized synthetic monophosphoryl lipid A(glycopyranoside Lipid A (GLA) adjuvant) was purchased from Avanti PolarLipids, Inc. (Alabaster, Ala.). Three mL 13 mm glass lyophilizationvials, caps and seals were from West Pharmaceutical Services (Lititz,Pa.). Concentrated 10× phosphate buffered saline (PBS), TWEEN 20, andsodium chloride were from Fischer Scientific (Fair Lawn, N.J.). Waterfor injection was purchased from Baxter Healthcare Corporation(Deerfield, Ill.). Peroxidase-conjugated affinipure donkey anti-mouseIgG (H+L) was from Jackson ImmunoResearch Laboratories, Inc. (WestGrove, Pa.). 3,3′,5,5′-tetramentylbenzidine (TMB) was from ThermoScientific (Rockford, Ill.).

HPV 16 L1 Capsomere Protein Purification

HPV 16 L1 capsomere protein was expressed in HMS174 E. coli containingthe plasmid HPV16-p3 grown in terrific broth. Cells were lysed by twopassages through a NIRO PANDA homogenizer at 800-1000 bar. The solubleportion was collected after centrifugation of cell lysate. Anionexchange was conducted by loading the soluble fraction onto a Q FASTFLOW column (GE Healthcare, Piscataway N.J.). The L1 protein, collectedin the flow through was then precipitated out using ammonium sulfateprecipitation at 30% saturation. The resuspended ammonium sulfateprecipitate was solubilized in a tris buffer and passed once through theNIRO PANDA homogenizer at—500 bar. The homogenate was then loaded onto aQ sepharose anion exchange column (GE Healthcare, Piscataway, N.J.) andthen eluted with a sodium chloride gradient. Collected fractionscontaining the L1 protein were exchanged into a 100 mM histidine bufferpH 7.1 by size exclusion chromatography.

Vaccine Formulation

Vaccines were formulated to contain 0.1 mg/mL HPV 16 L1 capsomere, 0 or0.5 mg Al/mL from ALHYDROGEL, 0 or 0.05 mg GLA/mL in 54 mM histidine HClpH 7.1 with 9.5 w/v % trehalose. Formulations were created to containcapsomere protein alone (protein), capsomere protein adsorbed toaluminum hydroxide (protein+alum) or capsomere protein adsorbed toaluminum hydroxide with GLA (protein+alum+GLA). Formulations wererotated end over end in 2 mL tubes for one hour to assure completeadsorption of protein to adjuvant.

Lyophilization

In certain examples, vaccines formulated with trehalose (othercarbohydrate agents can substitute for trehalose such as sucrose,chitosan etc.) were lyophilized with 1 mL of formulation per vial.Lyophilizer shelves were pre-cooled to −10° C. (FTS Systems Lyophilizer,Warminster, Pa.) and vials were placed on the shelves. Vaccineformulations were surrounded by vials filled with DI water to minimizeradiative heat transfer effects for vials near the edge of thelyophilizer shelves. The shelf temperature was decreased at a rate of0.5° C./min to −40° C. and then held at −40° C. for 1 hour to allow thesamples to completely freeze. Primary drying was initiated by decreasingthe chamber pressure to 60 mTorr and increasing the shelf temperature to−20° C. at a rate of 2° C./min. Samples were held at −20° C. for 20hours. Secondary drying was conducted by increasing the shelftemperature to 0° C. at a rate of 0.2° C./min, followed by an increaseto 30° C. at a rate of 0.5° C./min and holding the shelf temperature at30° C. for 5 hours. After drying, the shelf temperature was returned to25° C. and the chamber was back-filled with nitrogen until atmosphericpressure was reached. Chlorobutyl rubber stoppers were inserted intovials under a nitrogen atmosphere. Before storage at −80° C., vials weresealed with aluminum caps.

Elevated Temperature Incubation Study

To test the stability of vaccines at an elevated temperature, liquid andlyophilized vaccines were stored at 50° C. for 0 or 12 weeks. Time 0lyophilized vaccines refer to vaccines used immediately after removalfrom storage at −80° C.

Particle Size Analysis

Particles greater than 2 microns were measured by use of the FLOW-CAM(Fluid Imaging Technologies, Yarmouth, Me.). A 100 micron flow cell wasused at a flow rate of 0.08 mL/min with images taken at a rate of 10frames per second. A 10× objective and collimator were used. Light anddark settings of 17 and 15, respectively, were used to captureparticles. Formulations were diluted ten times for placebo formulations,and 100 times for formulations containing protein. A sample volume of0.35 mL was used for all formulations.

Differential Scanning Calorimetry (DSC)

Onset glass transition temperatures of placebo lyophilized formulationswere obtained using differential scanning calorimetry (Diamond DSC,Perkin Elmer, Waltham, Mass.). Triplicate samples were prepared insidean aluminum pan under dry nitrogen. Pans were cycled twice between 25°C. and 150° C. at a scan rate of 100° C./min. The second heating scanwas used to determine the onset glass transition temperature.

Transmission Electron Microscopy (TEM)

In other methods, vaccine or immunogenic formulations were adsorbed tocarbon-coated grids and negative stained with 2% uranyl acetate. Imageswere collected using a transmission electron microscopy. Samples ofvaccines containing one of each of the four capsomere types as well assamples of the tetravalent vaccine formulation that contained all fourtypes, were analyzed by TEM before and after lyophilization. Becausealuminum hydroxide microparticles can interfere with TEM analysis ofcapsomeres, samples tested with TEM did not contain aluminum hydroxide.In certain examples, vaccine formulations were adsorbed toformvar/carbon-coated, glow-discharged 400 mesh copper TEM grids. Aftersample adsorption, grids were washed with 5 mM EGTA and stained with1-2% uranyl acetate. Images were collected using a Philips CM10transmission electron microscope operating at 80 kV equipped with aGATAN BIOSCAN2 digital camera.

Size Exclusion Chromatography (SEC)

HPV 16 L1 capsomere protein was run on a SUPERDEX 200 INCREASE 10/300 GLcolumn (GE Healthcare Life Sciences) in a buffer containing 50 mM Tris,350 mM sodium chloride, 10% glycerol, 5 mM DTT at pH 8.1.

Fluorescence Melting Curve

Fluorescence melting curves were created to determine the proteinmelting temperature. Approximately 200 μL of 0.1 mg/mL HPV 16 L1capsomere was placed in a micro quartz cuvette. Fluorescence spectrawere collected from about 305 to 400 nm after being excited at 295 nm ona SLM Instruments Inc. fluorimeter (Urbana, Ill.). Spectra were recordedevery 5° C. from 20° C. to 90° C., after an equilibration time of tenminutes. Center of spectral mass calculations were used to create themelting curve.

Front Face Fluorescence

Three mL of vaccine formulation was placed in a quartz cuvette in afront face geometry holder with angle of incidence of 53° on afluorimeter. Samples were excited at 295 nm and the emission spectrumwas collected from 310 nm to 400 nm. The peak intensity at 331 nm fortime 0 samples and 340 for unfolded protein was monitored as acrylamidewas added. The Stern-Volmer constant was measured by solving thefollowing equation: Fo/F=1+Ksv[Q]. F0 is the fluorescence intensitywithout the quencher acrylamide, F is the fluorescence with the quencherpresent, Ksv is the Stern-Volmer constant and [Q] is the quencherconcentration. The maximum Ksv value of this setup was found using freetryptophan at 0.1 mg/mL and the maximum Ksv value for the HPV 16 L1capsomere was found by unfolding the protein overnight in 8M urea.

LI and V5 Epitope Binding Assay

To determine the conservation of the L1 and V5 capsomere epitopes, anELISA based assay was conducted. Vaccine formulations with and withoutaluminum hydroxide adjuvant were diluted in PBS such that 0.25, 0.125,0.0625, and 0 μg/well of HPV 16 L1 capsomere protein was coated on96-well Nunc flat bottom PolySorp Immuno plates and incubated overnightat 4° C. Plates were washed three times with 0.05% TWEEN 20 in PBS at300 μL/well. Plates were blocked with 100 μL/well of blocking buffer (5%dry milk, 0.05% TWEEN 20 in PBS) for 1 hour at 37° C. After blocking,blocking buffer was removed and primary antibodies, against either L1 orV5 at a dilution of 1:1000 in blocking buffer, were added 50 μL/well andincubated at 37° C. for 1 hour. After washing three times, secondaryantibody diluted 1:5,000 in wash buffer (0.05% TWEEN 20 in PBS) wasadded 50 μL/well and incubated at 37° C. for 1 hour. The secondaryantibody for L1 and V5 respectively was a goat anti-rabbit and a goatanti-mouse HRP conjugated IgG antibody. After washing five times, 50μL/well of Turbo TMB was added and plates were incubated at roomtemperature for five minutes. The reaction was quenched with 50 μL/well1 M sulfuric acid and plates were read for absorbance at 450 nm on aMolecular Devices Kinetic Microplate Reader (Sunnyvale, Calif.).

Vaccine Immunogenicity

Murine studies were conducted under the University of Colorado atBoulder Institutional Animal Care and Use Committee (IACUC) protocol#1209.02. Female Balb/c mice from Taconic (Hudson, N.Y.) were allowed toacclimate at least one week before use and were 10-11 weeks old at thestart of the immunization study. Mice had blood samples collected underisofluorane anesthesia on days 0, 21 and 36 through the retro orbitalcavity, and were injected intramuscularly on days 0 and 21 with variousformulations. Mice were injected with reconstituted lyophilized protein,protein+alum, protein+alum+GLA vaccines, and liquid GARDASIL andCERVARIX vaccines. Serum was separated by centrifugation at 10,000 rpmfor 14 minutes at 4° C. and stored at −80° C. until use.

Total Antibody Enzyme Linked Immunosorbent Assay (ELISA)

NUNC MAXISORB 96 well plates (Thermo Fischer Scientific, Rochester,N.Y.) were coated with 50 μL/well of 1 μg HPV 16 L1 capsomere/mL dilutedin PBS and incubated at 2-8° C. overnight. Plates were washed 3 timeswith PBS containing 0.05% TWEEN 20. Plates were blocked with 300 μL/wellof PBS with 1% BSA, incubated at room temperature for 2 hours, andwashed again. Serum was initially diluted in PBS with 1% BSA, 0.05%TWEEN 20, 100-fold for serum collected on days 0, 500-fold for serumcollected on day 14, and 1,000 or 5,000-fold for serum collected on Day28 for mice injected without and with adjuvant respectively. A series ofin-plate 2-fold dilutions were made for each sample. Plates wereincubated for 1.5 hours at room temperature and washed. Approximately 40μL of HRP-conjugated donkey anti-mouse antibody diluted 10,000 times wasadded to each well and incubated for 1.5 hours at room temperature withshaking, followed by washing. Approximately 40 μL TMB was added to eachwell and incubated for 15 minutes, followed by quenching with 40 μL of2N sulfuric acid. Plates were measured at 450 nm on a MOLECULAR DEVICESKinetic Microplate Reader (Sunnyvale, Calif.).

To determine titers, average OD 450 values as a function of dilutionwere fit to a 4-parameter logistic equation using SigmaPlot software.The constraints 0<min<0.15 and max<3.3 were used. A cutoff value of 0.5was used.

Pseudovirus Production

293TT cells were plated at a concentration of 7×106 cells/20 mL andallowed to adhere overnight. DNA plasmid for secreted alkalinephosphatase (SEAP), DNA plasmid for L 1 and L2 capsid proteins, andlipofectamine were incubated with OptiMEM-1 before being added to 293TTcells. Cells were incubated overnight with the DNA then harvested.TRITON-X, benzonase, plasmid safe, and ammonium sulfate were used tolyse cells. Pseudovirus was purified salt extraction, and collecting thesupernatant after centrifugation. Clarified cell lysate was added to anOPTIPREP gradient and separated by centrifugation. Fractions werecollected from the bottom of the gradient tube and assayed for DNA andprotein content by PICOGREEN assays and BCA assay, respectively.

Neutralizing Antibodies

293TT cells were grown, harvested, and counted. 100 μL/well of 3×105cells/mL were plated in 96 well tissue culture plates and incubated at37° C. for 2-5 hours. Pseudovirus was added to dilutions of mouse serumand incubated on ice for 1 hour. Approximately 100 μL ofpseudovirus/mouse serum solution was added to plated cells and incubatedat 37° C. for 3 days. After incubation, supernatant was collected fromcells. The GREAT ESCAPE SEAP Chemiluminescence test kit was used fordetection of SEAP. Plates were read on a luminometer at a setglow-endpoint of 0.20 seconds/well. The neutralization titer is definedas the dilution of mouse serum that neutralizes greater than 50% of thepseudovirus.

SDS PAGE and Western Blots

Pre- and post-lyophilization samples of vaccines containing aluminumhydroxide adjuvant as well as HPV16 L1 capsomeres, HPV18 L1 capsomeres,HPV31 L1 capsomeres, or HPV45 L1 capsomeres sampled prior tolyophilization and after lyophilization and reconstitution were analyzedusing Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDSPAGE). A similar analysis was conducted for samples of a tetravalentvaccine formulation containing aluminum hydroxide as an adjuvant and amixture of HPV16 L1 capsomeres, HPV18 L1 capsomeres, HPV31 L1 capsomeresand HPV45 L1 capsomeres. Samples were denatured by the addition ofSample Buffer (240mM Tris, 30% glycerol, 6% SDS, 6 mg/ml bromophenolblue and 15% P-mercapto ethanol [PMED and boiled at 95° C. for 10minutes. Samples were loaded with constant volume and run at 150V, 150mA for 1 hour and 10 minutes.

In certain examples, following electrophoresis, gels were placed inTransfer Buffer (250 mM Tris, 2M glycine, 20% methanol) for 20 minutesto remove SDS. Gels were transferred onto PVDF membrane for the Westernblot using a semi-dry transfer unit (Hoefer, Holliston, Mass. ) at 15Vfor 45 minutes. Following transfer, the blot was blocked in a 5% milksolution in Tris Buffered Saline with Tween 20 (TBST) (10 mM Tris, 150mM NaCl, 0.1% Tween 20) for one hour at room temperature. Primaryantibody diluted in TB ST was added (GARDASIL treated rat sera, 1:5000[HPV16 and 45]; a-HPV18 L1 mab specific for HPV18 L1, diluted 1:2000[Abcam, Cambridge, MA]; a-HPV313G11C8 specific for HPV31 L, diluted1:1000) and incubated with rocking at room temperature for one hour. Theprimary antibody was removed and the blot washed three times for 10minutes each with TBST. An appropriate secondary alkalinephosphatase-conjugated antibody (diluted 1:5000 is TBST) was then addedand incubated with rocking at room temperature for one hour. Thesecondary antibody was removed and the blot washed as before. Thecompleted blot was developed in an alkaline phosphate developer (250 mMTris, 250 mM NaCl, 12.5 mM MgCl2, 165 ug/ml 5-Bromo-4-chloro-3-indolylphosphate [BCIP], 22 ug/ml nitro blue tetrazolium [NBT]) until bandswere deemed sufficient. Blot was rinsed with deionized water to stop thedeveloping reaction.

In certain methods, aluminum hydroxide (alum)-adjuvanted RGI-VLP werelyophilized by lyophilizing with trehalose. Aliquots of a dry-powderformulation were incubated at 4° C., 20° C., 37° C. or 50° C. for either1 day, 1 week or 1 month, resuspended and used to immunize groups ofBalb/c (n=5) in a 3-dose regime (211 g VLP/dose; week 0/2/4; bloodfinally drawn at week 6). Immune sera were pooled for groups and testedby HPV16 L1-VLP and RGI-peptide ELISA, as well as L1- and L2-basedpseudovirion neutralization assays (LI- and L2-PBNA). Further, a T cellresponse was evaluated by IFNy ELISPOT using splenocytes that werepooled for groups.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of particular embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope herein. Morespecifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept as defined bythe appended claims.

What is claimed is:
 1. An immunogenic composition comprising: abroad-spectrum multi-targeted antigen construct; one or morenon-reducing disaccharide agents; one or more volatile salts; whereinthe immunogenic composition is essentially dried.
 2. The immunogeniccomposition according to claim 1, wherein the one or more non-reducingdisaccharide is selected from the group consisting of trehalose,sucrose, lactose, or combinations thereof.
 3. The immunogeniccomposition according to claim 1, wherein the one or more volatile saltscomprise one or more of ammonium acetate, ammonium formate, ammoniumcarbonate, ammonium bicarbonate, triethylammonium acetate,triethylammonium formate, triethylammonium carbonate, trimethylamineacetate trimethylamine formate, trimethylamine carbonate, pyridinalacetate and pyridinal formate, or combinations thereof.
 4. Theimmunogenic composition according to any one of the preceding claims,wherein the composition is stable for one month or greater at elevatedtemperatures.
 5. An immunogenic pharmaceutical composition of use as avaccine comprising: a construct composition according to claim 1 and apharmaceutically acceptable excipient.
 6. A method of preparing animmunogenic composition, the method comprising: (a) combining abroad-spectrum multi-targeted antigen construct with one or morenon-reducing disaccharide agents and one or more volatile salts in abuffer making a liquid immunogenic composition; (b) freezing the liquidimmunogenic composition; and (c) lyophilizing the frozen immunogeniccomposition creating an essentially dry powder of the immunogeniccomposition.
 7. The method according to claim 6, wherein the one or morenon-reducing disaccharide is selected from the group consisting oftrehalose, sucrose, lactose or combinations thereof
 8. The methodaccording to claim 6, wherein the one or more volatile salts compriseone or more of ammonium acetate, ammonium formate, ammonium carbonate,ammonium bicarbonate, triethylammonium acetate, triethylammoniumformate, triethylammonium carbonate, trimethylamine acetatetrimethylamine formate, trimethylamine carbonate, pyridinal acetate andpyridinal formate, or combinations thereof
 9. The method according toclaim 6, wherein the composition is stable for one month or greater atelevated temperatures.
 10. The method according to claim 6, wherein theone or more non-reducing disaccharide is trehalose, and the trehalose ispresent in a weight-to-volume concentration of from about 8% to about20% in the liquid vaccine formulation.
 11. The method according to claim6, wherein the freezing step comprises one of tray freezing, flashfreezing, lyophilization, shelf freezing, spray-freezing andshell-freezing.
 12. The method according to claim 6, wherein lyophilizedcomposition is reconstituted with an aqueous diluent to form areconstituted immunogenic composition.
 13. The method according to claim6, wherein the liquid immunogenic composition is prepared as ahypertonic mixture.
 14. The method according to claim 6, wherein thedried immunogenic composition is stored without refrigeration up to atemperature of about 40° C. to about 60° C.
 15. A method for elicitingan immune response to one or more pathogenic organisms in a subject, themethod comprising administering to the subject a reconstitutedimmunogenic composition according to claim 5 and eliciting an immuneresponse to one or more serotypes or types of pathogenic organisms inthe subject.
 16. A method for enhancing cross-reactivity of immuneresponses to a multi-targeted antigenic composition, the methodcomprising heating a lyophilized powder containing the multi-targetedantigen in a composition comprising, one or more non-reducingdisaccharide agents; and one or more volatile salts to a temperature ofabout 40 to about 60° C., administering the immunogenic compositionaccording to claim 5 and eliciting a cross-reactive immune response tomultiple serotypes or types of pathogens in the subject.